Specific Recognition of Macroscopic Objects by the Cell Surface: Evidence for a Receptor Density Threshold Revealed by Micrometric Particle Binding Characteristics  Stéphanie Sarda, David Pointu, Frédéric Pincet, Nelly Henry  Biophysical Journal  Volume 86, Issue 5, Pages 3291-3303 (May 2004) DOI: 10.1016/S0006-3495(04)74377-5 Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 1 CD19 receptors titration: the number of CD19 receptors per cell has been determined using increasing concentrations of FITC-labeled anti-CD19 and measuring fluorescence per cell using flow cytometry. The arbitrary values obtained were converted into bound anti-CD19 applying the autocalibration methods described in Materials and Methods and plotted as a function of antibody concentration (A). Experiments were performed at 4°C, in PBS buffer, pH 7.4. Cell concentration for these experiments was equal to 2.5×106 cells/ml. Here is a representative experiment of at least three separate titrations. Anti-CD19 binding analysis (B) was performed according to the Scatchard-like method (see Materials and Methods). The adjustment of the experimental points (•) to the analytical formula obtained (—) provided an association constant Ka equal to 8.2×108 M−1 and a mean number of sites equal to 4.1×105 receptors per cell. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 2 Flow cytometry biparametric dot-plots and histograms showing CD19 receptors distribution. LB cells were incubated at 4°C during 1h with 4μg/ml FITC-conjugated anti-cytokeratin (A and C) and with FITC-conjugated anti-CD19 (B and D). The fluorescence distribution of the labeled cells clearly displayed only one mode; it has been adjusted to a Gaussian distribution (—) of the form (1/σ2π)e−(x−x¯)2/2σ2, which provided a mean value of 47. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 3 Scattering (upper frames) and fluorescence (lower frames) biparametric dot-plots of interacting particles and cells. 1.5×105/ml LB cells were labeled (A, B, C) or not (D) with biotinylated anti-CD19 and put into contact with 1.5×106/ml streptavidin-coated particles. Flow cytometry data were acquired at various times after particle contact; (A) t=0, (B) t=1min, and (C and D) t=30min. Incubation was performed at 4°C, measurements at 20°C, all in PBS buffer, pH 7.4. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 4 Cells and particles interaction kinetic profile. The ratio of the number of cells having acquired enhanced fluorescence, i.e., located in the upper quadrant of the FL3 dot-plot (see Fig. 3), to the total number of living cells has been plotted as a function of time of contact with the particles. Cells had previously been labeled (●) or not (○) with biotinylated anti-CD19. Same experimental conditions as in Fig. 3. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 5 Optical (a) and fluorescence microscopy (b and c) pictures of labeled cells and particles sample. Fluorescence images of particles were recorded under epifluorescence lightening using a FITC (b) or a rhodamine (c) filter setup, showing the large spectra of the particles’ fluorescence. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 6 Binding sites distribution of particle-free and particle-bound cells. 5×105/ml LB cells labeled with biotinylated anti-CD19 were incubated with 5×106/ml particles for 60min. Once the binding process was achieved, cells with particles and cells without particles were physically sorted under magnetic field gradient. The cells in the supernatant, without particles, were then labeled with a saturating (1μg/ml) FITC-coupled streptavidin and their fluorescence histogram (dark shaded) recorded in the FL1 flow cytometer channel. The same labeling was performed in parallel on biotinylated cells before the incubation with the particles (open black). These are shown together with an unlabeled control cells histogram (dotted line); mean values in Table 1. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 7 Receptor density cutoff for the binding of a particle, obtained after subtraction of the particle-free cells distribution to the total cells distribution, normalization of each histogram to the same number of cells, and multiplication of each by their respective mean frequency, i.e., 0.4 and 0.6. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 8 Number of bound particles per cell: the cluster of the cells having bound particles (black frame) was selected on the biparametric dot-plot (A), at the kinetic plateau of the binding (same experimental conditions as in Fig. 2). Its fluorescence distribution was plotted according to a linear scale (B), which allowed resolving discrete populations of cells according to their bound-particles’ number. The fluorescent increment per bound particle was found equal to 17 arbitrary units. The number of cells per subpopulation (with 1, 2, 3, … , n particles) was counted and plotted as a function of n, the number of bound particles per cell (C). The experimental points (•) were adjusted to an exponential decay (—) of the form ν(n)=p×e−δ×n. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 9 Effect of particles/cells ratio. Cells and particles were put into contact as described in Fig. 3 except the particles/cells ratio was decreased. Ratios are equal to 2 (□), 7 (●), and 15 (♦). Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 10 Effect of the length of the molecular link. Cell/particle binding process was followed as in Fig. 3 except the molecular link presented by the cell was extended using a biotinylated (Fab)′2 fragment directed against the mouse anti-CD19 already bound on the B-cell (▴). Data obtained with the shortest link, i.e., biotinylated anti-CD19 (●). Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 11 Effect of stirrer rotation speed. The kinetics of the cell/particle binding process was followed as described in Fig. 3 for three different rotation speeds of the stirring setup: 5 RPM (□), 10 RPM (♦), and 20 RPM (●). Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 12 Distribution of the number of particles per cell. The number of cells, ni, in each subpopulation of cells bearing i particles was plotted as a function of i, for experiments performed with (■) or without (●) 0.1% sodium azide. The normalized frequency for a subpopulation of cells bearing i particles is equal to ni∑i=1nni×1νmax, where νmax is equal to the highest frequency of the sample. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 13 Geometry of the contact. This figure gives the parameters allowing the description of particle/cell contact, where rc is the radius of the contact area, rb is the radius of the particle, and h* is the penetration depth of the particle in the cell surface layer. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 14 Evaluation of cell-particle energy of adhesion. Cell and particle are pulled apart with micropipette. The various angles used for the calculation of the energy of adhesion are shown. Experiments were performed at room temperature. Biophysical Journal 2004 86, 3291-3303DOI: (10.1016/S0006-3495(04)74377-5) Copyright © 2004 The Biophysical Society Terms and Conditions